Abstract INTRODUCTION: In most Strongyloides stercoralis infected individuals, nematoidosis occurs asymptomatically, but in immunocompromised patients, it can cause hyperinfection. Serological techniques seem to be a good alternative for detecting this parasite. METHODS The frequency of seropositivity for strongyloidiasis in Alfenas, MG, was estimated using the enzyme linked immunosorbent assay on blood samples, between May and August of 2015. RESULTS: Out of 258 samples tested, 53.9% were positive, and the frequency of seropositive individuals was higher in the peripheral districts of the municipality. CONCLUSIONS: The results indicate high seropositivity rates for strongyloidiasis among the residents of Alfenas city.

SUMMARY Visceral leishmaniasis is a systemic and chronic disease and dogs are the main reservoir of the etiologic agent, Leishmania infantum (syn L. chagasi). A serological and molecular investigation of canine visceral leishmaniasis (CVL) was performed in the municipality of Alfenas, located in the southern region of Minas Gerais, where the disease is not endemic. Samples from 87 dogs were submitted to serological tests including the Dual Path Platform (DPP (r) ) CVL Bio-Manguinhos rapid test, an in-house enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT), as well as molecular techniques such as a conventional polymerase chain reaction (PCR) with the RV1/RV2 primers and a quantitative PCR (qPCR) with the LinJ31, Ldon and DNApol primers. Of the 87 serum samples, eight (9.2%) were positive for Leishmania using the DPP rapid test, but only four (4.6%) were confirmed by ELISA and two (2.3%) by IFAT. In these two serologically confirmed cases, spleen and liver samples were positive by all the employed molecular and parasitological procedures performed on spleen samples. When whole blood samples were used in the molecular assays, two samples (2.3%) were positive only by qPCR. DNA extracted and amplified from the spleens of seropositive dogs was sequenced, showing 100% of similarity with the Leishmania infantum (syn L. chagasi) sequence. Thus, the first cases of CVL have been confirmed in the Alfenas region, suggesting the importance of canine surveys in non-endemic municipalities for CVL to monitor disease progression and to prevent outbreaks.

Isolados de Cryptosporidium identificados em quatorze amostras de fezes, coletadas de cinco pacientes com infecção por HIV e de nove crianças imunocompetentes, residentes no estado de São Paulo, Brasil, foram submetidos a análise molecular por Nested-PCR, seguido da caracterização genética por polimorfismo do tamanho do fragmento de restrição (RFLP). A análise foi baseada na digestão, com a enzima de restrição RsaI, de um fragmento de DNA amplificado do gene que codifica a proteína de parede do oocisto de Cryptosporidium (COWP). Baseado nesta análise, quando comparadas aos padrões utilizados, quatro amostras foram identificadas como Cryptosporidium parvum, oito como Cryptosporidium hominis e duas apresentaram um perfil correspondente ao de Cryptosporidium meleagridis. O uso de métodos moleculares pode ser útil para identificar a fonte das infecções e os fatores de risco relacionados à infecção por Cryptosporidium em nossas comunidades.

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Considerando a escassez de antígenos quimicamente definidos, realmente úteis e confiáveis para aplicação na soroepidemiologia da esquistossomose em larga escala, foi proposto, neste trabalho, um método alternativo para a seleção de clones de cDNA que expressam proteínas com putativo potencial diagnóstico na esquistossomose. Empregando anticorpos específicos contra uma fração proteica de 31/32 kDa (Sm31/32), purificados através da dissociação de imunocomplexos, foram selecionados cinco clones de cDNA a partir de genoteca de verme adulto de Schistosoma mansoni. O seqüenciamento parcial destes clones demonstrou que todos eram relacionados ao S. mansoni: dois apresentaram homologia com a proteína de choque térmico de 70 kDa e os demais com glutationa S-transferase, "homeodomain protein" e uma etiqueta de seqüência expressa (EST). Este último foi o clone que melhor reagiu, durante o processo de seleção, com os anticorpos anti-Sm31/32 dissociados de imunocomplexos. Baseado na seqüência de aminoácidos deste clone, dois peptídeos foram quimicamente sintetizados e analisados separadamente frente a misturas de soros de indivíduos normais e de pacientes com esquistossomose mansoni. Ambos os peptídeos demonstraram uma intensa reatividade somente contra a mistura de soros positivos, sugerindo que estes peptídeos podem ser úteis como antígenos para o diagnóstico da esquistossomose mansoni.

Considering the scarcity of defined antigens, actually useful and reliable for use in the field studies, we propose an alternative method for selection of cDNA clones with potential use in the diagnosis of schistosomiasis. Human antibodies specific to a protein fraction of 31/32 kDa (Sm31/32), dissociated from immune complexes, are used for screening of clones from an adult worm cDNA library. Partial sequencing of five clones, selected through this strategy, showed to be related to Schistosoma mansoni: two were identified as homologous to heat shock protein 70, one to glutathione S-transferase, one to homeodomain protein, and one to a previously described EST (expressed sequence tag) of S. mansoni. This last clone was the most consistently reactive during the screening process with the anti-Sm31/32 antibodies dissociated from the immune complexes. The complete sequence of this clone was obtained and the translation data yielded only one ORF (open reading frame) that code for a protein with 57 amino acids. Based on this amino acid sequence two peptides were chemically synthesized and evaluated separately against a pool of serum samples from schistosomiasis patients and non-schistosomiasis individuals. Both peptides showed strong reactivity only against the positive pool, suggesting that these peptides may be useful as antigens for the diagnosis of schistosomiasis mansoni.

The high sensitivity and the ability to diagnose schistosomiasis in a very early phase after infection have indicated the detection of IgM antibodies to Schistosoma mansoni gut antigens by the immunofluorescence test (IgM-IFT) as a useful serological test for epidemiological studies in low endemic areas. When applied in a follow-up study for two years, higher rates of seroconversion from IFT negative to positive were observed during the summer months, suggesting seasonal transmission of schistosomiasis in the rural area of the municipality of Itariri (São Paulo, Brazil). In each survey, blood samples from about 600 schoolchildren were collected on filter paper and submitted to IgM-IFT. When the blood samples were classified for the IgM antibody levels, according to the intensity of fluorescent reaction observed at fluorescence microscopy, and correlated to the egg counts in the Kato-Katz positive patients, no association was observed. This observation might suggest that the intensity of fluorescence observed in the IgM-IFT, as an indicator of IgM antibody levels, could not be an useful seroepidemiological marker for classifying areas of low endemicity according to degrees of infection.

O potencial diagnóstico dos anticorpos IgM e IgA contra antígenos do tubo digestivo do Schistosoma mansoni, detectados através da reação de imunofluorescência indireta utilizando-se cortes parafinados de vermes adultos, foi avaliado, comparativamente aos resultados do exame parasitológico de fezes, para fins epidemiológicos em áreas de baixa endemicidade para a esquistossomose. Amostras de sangue em papel de filtro foram coletadas de escolares, residindo em duas localidades diferentes dentro do município de Itariri (São Paulo, Brasil), com características epidemio-lógicas distintas em relação à esquistossomose. Os dados parasitológicos e sorológicos foram comparados aos obtidos com um outro grupo de escolares, residentes em uma área não endêmica para esquistossomose. Os resultados demonstraram a falta de sensibilidade do método parasitológico para detecção de indivíduos com baixa carga parasitária e indicam a potencialidade do método sorológico como importante instrumento a ser incorporado aos programas de controle e de vigilância da esquistossomose, para verificação da real situação da esquistossomose em áreas de baixa endemicidade.

The diagnostic potential of circulating IgM and IgA antibodies against Schistosoma mansoni gut-associated antigens detected by the immunofluorescence test (IFT) on adult worm paraffin sections was evaluated comparatively to the fecal parasitological method, for epidemiological purposes in low endemic areas for schistosomiasis. Blood samples were collected on filter paper from two groups of schoolchildren living in two different localities of the municipality of Itariri (São Paulo, Brazil) with different histories and prevalences of schistosomiasis. The parasitological and serological data were compared to those obtained for another group of schoolchildren from a non-endemic area for schistosomiasis. The results showed poor sensitivity of the parasitological method in detecting individuals with low worm burden and indicate the potential of the serological method as an important tool to be incorporated into schistosomiasis control and vigilance programs for determining the real situation of schistosomiasis in low endemic areas.

Uma biblioteca de expressão de cDNA de verme adulto de S. mansoni foi selecionada utilizando-se soros de macacos babuínos em fase inicial da infecção. Os clones que reagiram positivamente com os soros de infecção recente foram examinados quanto à reatividade contra soros normais e soros de infecção heteróloga. Com a finalidade de se conseguir melhor discriminação entre reatividade positiva com o anticorpo específico e aquela devido aos anticorpos anti-E. coli residuais, um clone de cDNA não relacionado ao S. mansoni foi plaqueado em mistura com o clone positivo. O plano de fundo negativo proporcionado pelo clone não relacionado forneceu o contraste necessário para discriminar a reação positiva específica. Esta metodologia de caracterização dos clones de expressão permite alcançar rapidamente os clones que apresentam o perfil de reatividade desejada, para posterior sequenciamento, subclonagem e expressão

A S. mansoni adult worm cDNA expression library was screened with sera from baboons in a early phase after infection. The clones that were positive with the early infection sera were examined for reactivity with pre-infection sera and heterologous infection sera. In order to discriminate a positive antibody reaction from the reactivity due to residual anti-E. coli antibodies, an unrelated cDNA clone was plated with the positive clone. The unrelated clone provided the negative background and the contrast necessary to discern a positive antibody reaction. In this way, we were able to eliminate selected clones that were positive with the pre-infection sera or heterologous infection sera. This characterization of the expression library clones enabled us to quickly target only clones with the desired pattern of antibody reactivity for sequencing, subcloning, and expressing

Em pacientes com esquistossomose, são encontrados anticorpos contra grande número de antígenos parasitários, e aqueles contra formas evolutivas jovens do parasita demonstraram que eram eficientes marcadores imunológicos para o diagnóstico da esquistossomose. Padrões de queda de anticorpos IgM e IgG contra cercária e esquistossômulo foram aqui estudados, comparativamente aos dos anticorpos contra verme e ovo, em pacientes esquistossomóticos após quimioterapia, abordando aspectos soroepidemiológicos. Dados obtidos no estudo de 359 amostras de soros, pertencentes a pacientes infectados por Schistosoma mansoni, indivíduos não infectados e pacientes acompanhados pós-tratamento por um período de 12 a 15 meses, forneceram base para postular padrão geral da cinética de queda de anticorpos. Antes do tratamento, o padrão de anticorpos é representado por curva unimodal, que se modifica para curva bimodal após tratamento, e finaliza com curva unimodal semelhante à do grupo de indivíduos não infectados. Diferentes tipos de anticorpos foram classificados em quatro categorias, conforme características apresentadas na queda, e o anticorpo IgM anti-esquistossômulo foi considerado como pertencente à categoria de anticorpos com queda moderada, enquanto que os demais anticorpos contra formas evolutivas jovens do parasita, à categoria daqueles com queda lenta. A metodologia aqui empregada permite identificar anticorpos potencialmente mais adequados a serem detectados em programas para controle da esquistossomose ou de outras parasitoses no campo

Antibodies to a number of parasite antigens are found in schistosomiasis patients, and antibodies to early developmental stages were demonstrated to be efficient immunologic markers for the diagnosis of schistosomiasis. In the present study, decay patterns of IgM and IgG antibodies against cercariae and schistosomula were investigated, in comparison to antibodies against worms and eggs in schistosomiasis patients after chemotherapy, for an investigation of seroepidemiologic aspects. Data obtained in the study of 359 serum samples from patients with Schistosoma mansoni infection, noninfected individuals, and patients followed-up for a period of 12 to 15 months after treatment provided the basis to postulate a general pattern for the kinetics of antibody decay. Before treatment, the antibody pattern was represented by a unimodal curve, which shifted to a bimodal curve after treatment, and ended with a unimodal curve similar to that for the noninfected group. Different types of antibodies were classified into four categories according to their decay features, and anti-schistosomulum IgM was classified into the moderate-decay caterogy, whereas other antibodies to early parasite stages were classified into the slow-decay category. The present methodology permits the identification of the most suitable antibodies to be detected in field control programs for schistosomiasis or other parasitoses